INTRODUCTION:

Ambroxol hydrochloride chemically 4-[[(2-amino-3, 5-dibromophenyl)-methyl]-amino]-cyclohexanol is an active metabolite of bromhexine, is an mucolytic expectorant particularly useful in bronchitis with chronic obstructive bronchitis and more effective in silicosis as secretolytic and surfactant stimulant asthama, sinusitis secretory otitis media, smokers cough if mucus plugs are present¹. It increases the amount of antibiotic penetration and thus reduces daily dose of gatifloxacin and exhibits anti-inflammatory properties². Ambroxol is used for the treatment of upper respiratory tract infection for adults.

Thorough survey of literature on ambroxol revealed that, its quantitative determination attracted workers in field because of its physiological significance and many physical methods viz., spectrophotometric method^3-9, RP-HPLC¹⁰ and HPLC and chemometrics-assisted UV-spectroscopy method¹¹have been applied to accomplish the purpose.

However methods on spectrophotometric determination of this drug involving ion pair complexes with common and versatile acidic dyes viz., bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol purple (BCP) are not reported yet. This prompted the authors to develop extractive spectrophotometric methods for the determination of ambroxol hydrochloride using the above mentioned dyes.

In this paper we report three simple and sensitive extractive spectrophotometric methods for the assay of ambroxol hydrochloride. The methods are based on ion-pair complexation of drug with dyestuffs such as bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol purple (BCP) and subsequent extraction into chloroform and measure the absorbance of colour complex.

EXPERIMENTAL:

Ambroxol hydrochloride is procured from Hetero labs limited, Hyderabad as a gift sample. The dyestuffs viz., BTB, BPB and BCP (AR grade) supplied by SD Fine Chemicals Ltd. Mumbai, are used without any further purification. The dyestuffs were used as 0.05% solutions in doubly distilled water. Sodium acetate-hydrochloric acid buffers¹² of pH 2.5, 2.8 and 3.5 were prepared by mixing 50ml of 1.0M sodium acetate solution with 50.50, 49.50 or 46.25 ml respectively, of 1.0 M HCl solution and diluted to 250 ml with doubly distilled water. The pH of each solution was adjusted to an appropriate value with the aid of a pH meter. Chloroform (HPLC grade) supplied by SD Fine Chemicals Ltd. Mumbai is used throughout the work. Stock solutions were prepared for all the dyes and drugs (25mg/100ml).

The spectra (Fig. 1) of ion-pair complexes have been recorded on SHIMADZU 140 double beam spectrophotometer, Thermo Nicolet 1000 and also on ELICO 159 UV-Visible single beam spectrophotometer using quartz cells of 10 mm path length. An Elico model Li-120 pH meter was used for pH measurement.

Fig. 1. Absorption spectra of Ambroxyl hydrochloride-dye complex extracted into 10 ml chloroform:

(a) drug = 25 mg ml^-1 + 5 ml of 0.05% BPB + 5 ml of pH 2.5 buffer; (b) drug = 16.0 mg ml^-1 + 5 ml of 0.05% BTB + 5 ml of pH 2.8 buffer; (c) drug = 35.0 mg ml^-1 + 5 ml of 0.05% BCP + 5 ml of pH 3.5 buffer.

Calibration curve:

Different aliquots of drug solution were transferred into 125 ml separating funnel. To this 5 ml of buffer (pH 2.5, 2.8 and 3.5), 5 ml of dye were added and total volume was made up to 20 ml with water. 10 ml of chloroform was added and the contents were shaken for 5 min. The two layers were allowed to separate for 5 min. The organic layer was separated and absorbance of yellow colored solution which is stable at least for 3 hrs is measured at 414, 412 and 407nm against blank similarly prepared. The same procedure of analysis is followed either for assay of pure drug or for dosage form. The calibration graphs (Fig. 2a, 2b, 2c) are linear over the concentration ranges are within the permissible range. The optical characteristics and statistical data for the regression equation of the proposed methods are presented in (Table 1).

Procedure for the assay of pure drug

Six different solutions of pure drug in the range of calibration curve were selected and the recovery experiments were performed. The recoveries and their relative standard deviations are tabulated in (Table 2).

Fig. 2a Calibration graph for Drug-BTB ion pair complex

Fig. 2b Calibration graph for Drug-BPB ion pair complex

Fig. 2c Calibration graph for Drug-BCP ion pair complex

Procedure for the assay of dosage forms

Ten tablets of Mucolite-3 mg are powdered and dissolved in doubly distilled water and stirred thoroughly, filtered through a Whatman No. 42 filter paper. This solution was transferred into 100 ml standard volumetric flask and diluted with doubly distilled water as required. Different solutions of drug in the range of calibration curve were chosen and the assay was estimated using the calibration curve. The results of the recovery experiments are tabulated in (Table 3).

TABLE -1: OPTICAL CHARACTERISTICS AND STATISTICAL FOR THE REGRESSION EQUATIONOF THE PROPOSED METHODS

Parameters

Extraction methods with

BTB

BPB

BCP

λ_max (nm)

Beer’s law limit (μg ml^-1)

Molar absorptivity (L mol^-1 cm^-1)

Formation constant, K,M^-1

Sandell sensitivity (μg cm^-2)

Slope (specific absorptivity), b

Intercept (a)

Correlation coefficient (r)

Standard deviation of intercepts (% n=6)

Limit of detection, μgml^-1

Limit of quantification, μgml^-1

Regression equation

414

2.5 - 25

15481

5.26 x 10⁵

0.0258

0.0387

-0.0043

0.997

0.00625

0.5326

1.5978

Y= 0.0387C- 0.0043

412

4.0 - 30

12432

3.7 x 10⁵

0.0533

0.0188

0.0219

0.997

0.0035

0.622

1.866

Y= 0.0188C +0.0219

407

4.0 - 40

8842

2.5 x 10⁵

0.033

0.0299

0.0021

0.999

0.0031

0.3413

1.024

Y=0.0299C +0.0021

^aWith respect to Y=bc+a, where C is the concentration (μg ml^-1) and Y is absorbance

^bSix replicate samples.

TABLE – 2: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF AMBROXOL HYDROCHLORIDE IN PURE FORM

Taken (μg ml^-1)	Proposed methods						Reference method [7]
	Found (μg ml^-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCP	BTB	BPB	BCP	Recovery (%)
3 6 9 12 15 RSD (%)	2.97 6.03 9.02 12.03 15.02	2.99 6.03 9.06 11.95 15.03	3.03 6.003 9.04 11.98 14.98	99.16 100.44 100.29 100.22 100.2 0.511	99.77 100.45 100.68 99.59 100.23 0.455	101.09 100.05 100.44 99.81 99.88 0.527	99.88 0.381
Mean±SD t-test F-test				100.05±0.51 0.67 1.8	100.14±0.45 1.1 1.43	100.25±0.53 1.38 1.93	99.88±0.381

TABLE – 3: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF AMBROXOL HYDROCHLORIDE IN PHARMACEUTICALS FORM

Taken (μg ml^-1)	Proposed methods						Reference method [7]
	Found (μg ml^-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCP	BTB	BPB	BCP	Recovery (%)
Mucolite 3mg/tablet 3.5 7 10.5 14 17.5 RSD (%)	3.49 6.96 10.49 13.98 17.52	3.47 6.99 10.5 13.97 17.49	3.56 6.96 10.51 14.01 17.45	99.85 99.39 99.97 99.9 100.14 0.279	99.31 99.87 100.04 99.82 99.94 0.284	101.87 99.53 100.1 100.1 99.69 0.935	99.27 0.808
Mean±SD t-test F-test				99.85±0.28 1.98 0.12	99.8±0.28 1.78 0.125	100.24±0.94 1.96 1.36	99.27±0.81

RESULTS AND DISCUSSION:

Ambroxol hydrochloride forms ion-pair complexes in acidic buffer with dyestuffs such as bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol purple (BCP) and these complexes are quantitatively extracted into chloroform. Ion-pair complexes of drug with BTB, BPB and BCP absorbed maximally at 414, 412 and 407 nm, respectively. The reagent blank under similar conditions showed no absorption.

In order to establish molar ratio between ambroxol hydrochloride and dyestuffs used, the Job’s method of continuous variation¹³ has been applied. In this method, solutions of drug and dyestuff with identical molar concentrations (2 x 10^-4M) were mixed in varying volume ratios in such a way that the total volume of each mixture was the same. The absorbance of each solution was measured and plotted against the mole fraction of the drug, [drug]/ [drug] + [dyestuff] (Fig. 3). This measurement showed that 1:1 complex was formed with each dyestuff. The formation constants^14,15 were also estimated and found to be 5.26x 10⁵, 3.7x 10⁵ and 2.5x 10⁵K M^-1for complexes with BTB, BPB and BCP respectively.

Ambroxol hydrochloride contains secondary amino group which is protonated in acid medium, while sulphonic acid group is present in BTB, BPB and BCP, that is the only group undergoing dissociation in the pH range 1-5. The colour of such dyes is due to the opening of lactoid ring and subsequent formation of quinoid group. It is supposed that the two tautomers are present in equilibrium but due to strong acidic nature of the sulphonic acid group, the quinoid body must predominate. Finally the protonated ambroxol hydrochloride forms ion-pairs with the dyestuffs which are quantitatively extracted into chloroform. The possible reaction mechanisms are proposed and given in (Scheme 1).

Fig. 3 Continuous-variations study of drug-dye systems: [Drug] = [Dye] = 2x10^-4M

The influence of pH on the ion-pair formation of ambroxol hydrochloride with various dyestuffs has been studied using sodium acetate-hydrochloric acid buffer. The results are shown in (Fig. 4). It is evident that absorbance of complexes with BTB, BPB and BCP was found to be constant within the pH ranges 2.2-3.3, 2.0-3.0 and 2.8-3.8 respectively. Thus, all the absorbance measurements were made at pH 2.8, 2.5 and 3.5 with BTB, BPB and BCP respectively.

Fig. 4 Effect of pH

[Drug] = [20µg ml^-1, [Dye] = 5ml of 0.05%

The effect of dyestuff concentrations was also studied by adding different volumes of dyestuff to a constant amount of ambroxol hydrochloride (20 µg ml^-1). It is apparent from Fig. 5 that the maximum absorbance, in each case, was found with 2.0 ml of dyestuff, beyond which absorbance was constant. Thus, 5 ml of each dyestuff was used for ion-pair formation throughout the experiment.

Fig. 5 Influence of the volume of 0.05% Dye

[Drug] = [20µg ml^-1]

A systematic study of the effect of foreign species present along with ambroxol hydrochloride on the determination of ambroxol hydrochloride at 20 µg ml^-1 level was undertaken. This study was carried out by following the proposed procedures for a 10 ml sample system, by adding a known amount of foreign species to an ambroxol hydrochloride solution of 20 µg ml^-1. Table 4 summarizes the results obtained. However, the drug content from the powdered tablets was extracted into chloroform, which completely removes any interference by the common excipients found in formulations.

TABLE – 4: INTERFERENCE STUDY

S. No.

Excipients

Tolerance limit

(μg ml^-1)

Microcrystalline cellulose

Starch

Lactose

Magnesium stearate Colloidal

silicon dioxide

Titanium dioxide

165

125

Validation of the proposed method:

All the three proposed methods have been validated in terms of guideline proposed by ICH¹⁶ viz. selectivity, specificity, accuracy, precision, limits of calibration curve, LOD, LOQ, robustness, ruggedness and regression equation. The student t-test and variance F-test have been performed in comparison with a reference method. Table 1 summarizes the values for Beer’s law limits, molar absorptivity, regression equation, correlation coefficients, relative standard deviation and recoveries. To test the reproducibility of the proposed methods, six replicate determinations of 20µg ml^-1of ambroxol hydrochloride were made. The coefficient of variation was found to be less than 1.2% for all the procedures.

The proposed methods have been successfully applied to the determination of ambroxol hydrochloride in pharmaceutical preparations. The performance order of the proposed methods is BTB>BPB>BCP. The results obtained and shown in Table 2 and Table 3 were compared to those obtained by a reference method⁷ by means of t-test at 95% confidence level. In all cases, the average results obtained by proposed methods and reference method were statistically identical, as the difference between the average values had no significance at 95% confidence level.

The proposed methods are simple, sensitive and reproducible and can be used for routine analysis of ambroxol hydrochloride in pure form and in formulation.

CONCLUSIONS:

Ambroxol hydrochloride formed ion pair complexes with acidic dyes with 1:1 composition and extractable in chloroform for assay of drug. The method is validated and applied to pharmaceuticals.

ACKNOWLEDGEMENTS:

The authors are grateful to Head, Department of Chemistry and Principal, Nizam College for providing facilities. MC is thankful to UGC for FDP fellowship. TV is thankful to the Management of SAP College, Vikarabad for providing facilities and to the UGC for financial assistance under Major Research Project.

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Received on 26.04.2011 Modified on 22.05.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1109-1113